Often molecules have to be adsorbed onto a surface in a localised way. Examples hereof are found in the fields of sensors where in arrays chemical probes or sensitisers are deposited, or in the emerging field of nanotechnology chemical functionality etc.
Techniques for localised deposition or absorption of (bio)molecules include but are not limited to classical lithography (etching or lift-off), direct use of UV light through metal mask or e-beam or laser ablation, soft lithography techniques (micro contact printing, imprinting), micro fluidics, the use of light addressable/activated molecules with lasers/scanning light source like e.g. laser, liquid spotting, . . . . See for instance table 1 of International Patent Application WO 01/84234 and Blawas (1998, Biomaterials 18:595-609). These techniques are well known to a person skilled in the art.
There are several drawbacks or disadvantages coupled to the currently available techniques. These include among others contamination with resists or with solvents used to remove resists, the possible damaging effect of residues to subsequently deposited molecules (e.g. biomolecules sensitive to the presence of organic solvents), the need for intricate/expensive equipment or handling to actually create a patterned chemical surface (e.g. lithography tools, spotting). Micro contact printing, although a simple technique in itself, encounters problems with printing polar moieties and with proper alignment, spotting limited resolution/pitch, etc. In general, expensive tools and equipment are needed.
Adsorption entails physisorption or chemisorption of molecules whereby the latter is preferred from robustness point of view, the possibility of re-use and stability. Examples hereof include in situ synthesis of oligonucleotides on chips, chemisorption via labelled molecules/probes with half of a reactive couple, in situ activation such as NHS/EDC coupling etc.
Possibly, labelling of the molecules can be done before deposition, whereby ex situ purification processes can be used to reduce the deposition process to a one-step process. Efficiency of deposition is often limited with in situ synthesis techniques and/or chemical activation being multi-step processes for adsorption.
There are several methods described in the art that relate to deposition of molecules on a surface and to checking of such binding or deposition. Published US Patent application US 2003/0059807 (Roach et al.), for instance, describes a method for detecting specific interactions between molecules through measuring the heat of binding generated when specific binding pairs interact with each other. The binding event can consist of hybridisation between complementary nucleic acids, but may also consist of other interactions between molecules such as protein/protein, peptide/protein, antigen/antibody (Ag/Ab), protein/DNA interactions or the like. The devices disclosed in Roach et al. measure the heat of binding through arrays of thermistors, possibly integrated in a microelectronic chip or in a multi-well microtiterplate.
International patent application WO 01/84234 (Pieken et al.) describes a novel chemoselective method for immobilising molecules (in particular biomolecules) on a support using cycloaddition reactions such as a Diels-Alder reaction. The support is preferably glass or controlled pore glass. Chemoselectivity is obtained by the chemical nature of the diene/dienophile.
Yousaf and Mrkisch (1999, J. Am. Chem. Soc 121:4286-4287) have described the immobilisation of proteins to electroactive self-assembled monolayers (SAMs) that present a quinone group. In a first step, a mixed monolayer presenting a hydroquinone (HQ) group undergoes oxidation at 220 mV to a quinone (Q) group (and reduction at −150 mV). In a next step, cyclopentadiene (cp), present in an electrolyte solution, reacts with Q via a Diels-Alder reaction.
The Diels-Alder reaction is just one example of a method for bio-immobilisation, well-suited for tailoring monolayers with peptides, carbohydrates and low-molecular weight ligands. This method allows quantitative attachment of groups in low densities and for sequential immobilisation of several ligands to a common substrate (with independent control over the density). Moreover, this method allows for immobilisation of active ligands that can be turned on at discrete times.
Diels-Alder reactions, advantageously, can be carried out in aqueous phase, the Diels-Alder reaction is tremendously accelerated in water and is very fast at room temperature or slightly below.
Published US Patent application US 2002/0051788 (Pozsgay) describes a method for covalently linking biomolecules under neutral conditions using a Diels-Alder reaction. Spontaneous binding between both molecules was here obtained. It was observed that described method provides a degree of control over the rate of coupling between the diene and the dienophile, by modification of the linking moieties. The reaction occurred preferably at or about room temperature.